Investigation Title Transcription profiling of purified populations of normal human breast luminal and myoepithelial cells
Comment[Submitted Name] BBC-LumMyo1
Experimental Design reference_design cell_type_comparison_design transcription profiling by array
Experimental Design Term Source REF mo mo EFO
Comment[ArrayExpressReleaseDate] 2004-05-04
Comment[AEMIAMESCORE] 4
Comment[ArrayExpressAccession] E-MEXP-36
Comment[MAGETAB TimeStamp_Version] 2011-06-30 21:38:21 Last Changed Rev: 14857
Experimental Factor Name cell_type
Experimental Factor Type cell_type
Experimental Factor Term Source REF
Person Last Name Grigoriadis
Person First Name Anita
Person Mid Initials
Person Email Anita.Grigoriadis@icr.ac.uk
Person Phone 00 44 2076799055
Person Fax
Person Address Insitute of Cancer Research, 237 Fulham Road, London, SW3 6JB, U.K.
Person Affiliation Breakthrough Breast Cancer Centre
Person Roles submitter
Person Roles Term Source REF mo
Quality Control Type dye_swap_quality_control
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2004-05-04
PubMed ID 15126339
Publication DOI 15126339
Publication Author List Jones, Chris; Mackay, Alan; Grigoriadis, Anita; Cossu, Antonio; Reis-Filho, Jorge S.; Fulford, Laura; Dexter, Tim; Davies, Susan; Bulmer, Karen; Ford, Emily; Parry, Suzanne; Budroni, Mario; Palmieri, Giuseppe; Neville, A. Munro; O'Hare, Michael J.; Lakhani, Sunil R.
Publication Title Expression Profiling of Purified Normal Human Luminal and Myoepithelial Breast Cells: Identification of Novel Prognostic Markers for Breast Cancer
Publication Status journal_article
Publication Status Term Source REF mo
Experiment Description Purified populations of normal human breast luminal and myoepithelial cells were compared with cDNA microarray analysis.
Protocol Name P-MEXP-981 P-MEXP-976 P-MEXP-975 P-MEXP-1545 P-MEXP-972 P-MEXP-969 P-MEXP-968 P-MEXP-971 P-MEXP-977
Protocol Type grow grow grow specified_biomaterial_action nucleic_acid_extraction labeling labeling hybridization bioassay_data_transformation
Protocol Description All cells were cultured in an atmosphere of 5% (v/v) carbon dioxide in a humidified incubator at 36.5 C. Cells at 70% confluency were fed the day before lysis. RPMI 1640; 10% FCS; 1.25 ug/ml Insulin; 1.25 ug/ml Hydrocortizone; 100 ng/ml Cholera Toxin; 1% Penicillin/Streptomycin (10000 units). All cells were cultured in an atmosphere of 5% (v/v) carbon dioxide in a humidified incubator at 36.5C. Cells at 70% confluency were fed the day before lysis. Ham's f-12; 10% FCS; 5 ug/ml Insulin; 1 ug/ml hydrocortizone; 10 ng/ml human recombinant EGF; 1% Penicillin/Streptomycin (10000 units); 100 ng/ml Cholera Toxin. All cells were cultured in an atmosphere of 5% (v/v) carbon dioxide in a humidified incubator at 36.5C. Cells at 70% confluency were fed the day before lysis. Luminal-myoepithelial cell separation
Fresh reduction mammoplasty (cosmetic surgery) human tissue was collected with informed consent. On transfer to the laboratory 150-200 g of fibro-fatty tissue was cut up, using scissors, into small fragments (2-5 mm). Fragments were transferred to 500 ml of Leibovitz L-15 medium plus 5% FCS, penicillin (100 IU/ml), streptomycin (100 ug/ml), gentamycin (50 ug/ml) and Fungizone (amphotericin B, 2.5 ug/ml), in a glass bottle with a magnetic flea, and type 1 collagenase (Sigma) added to final concentration of 0.05 mg/ml. The tissue was digested, with stirring, for 18-24 hrs at 37C, following which the stirrer was switched off and the digest allowed to settle for 20-30 min at 37C. The upper fat layer was removed and the remainder spun down. The pellet, consisting of partly digested epithelial fragments (organoids) was resuspended in 100 ml L-15 medium plus 5%FCS, penicillin (100 IU/ml), streptomycin (100 ug/ml), gentamycin (50 ug/ml) and Fungizone (amphotericin B, 2.5 ug/ml) and allowed to settle overnight at 4C. Sedimented fragments were collected by aspiration and subjected to a further treatment with 0.25 mg/ml collagenase type 1 for 1-2 hr until organoids were completely free from attached stroma. They were then spun down, resuspended in fresh medium and filtered successively through 140-micron and 53-micron nylon monofilament meshes. Organoids trapped on the mesh were collected by back-flushing and pooled.
Purified organoids were then plated in 180-cm2 Nunc plastic tissue culture flasks in RPMI1640 medium plus 10% (v/v) FCS and penicillin/streptomycin, containing 5 ug/ml bovine insulin, 5 ug/ml hydrocortisone and 20 ng/ml cholera toxin (all from Sigma). Cultures were maintained at 37C with 5% CO2/air gas phase until the organoids and attached and mobilised (7-10 days).
Cultures were then washed (x2) with sterile Ca++/Mg++-free phosphate buffered saline PBS and refed with a Ca++-free medium overnight (Joklik's modification of Dulbecco's Eagle's medium plus 10% dialysed FCS and penicillin/streptomycin). The following day the supernatant, which contained detached cells enriched for luminal epithelial cells, was removed and spun down. Cultures were reffed with RPMI1640/FCS medium.
The cell pellet was resuspended in 1 ml of L-15/10%FCS medium containing 5 ug/ml ICR-2 rat monoclonal antibody (Hybridoma Unit, Institute of Cancer Research, Sutton, Surrey) plus 5 ug/ml each of mouse anti-CD10 (DAKO) and mouse anti-beta4 integrin (Santa Cruz) antibodies and incubated over ice with rocking for 1 hr. Cells were then pelleted and washed (x2) with serum-free L-15 medium and next resuspended in 0.5 ml L-15 medium plus 10% (v/v) FCS and 200 ul of goat anti-rat antibody conjugated MACS superparamagnetic MicroBeads (Miltenyi). After 30 min incubation on ice, cells were pelleted by centrifugation, resuspended in L-15 medium and applied to a pre-washed BS-series stainless steel mesh column in the Miltenyi VarioMACS magnet system. After loading, unattached cells were washed through the column using L-15 medium, and the column was then removed from the magnet and the retained cells flushed out. After pelleting by centrifugation and resuspension in L-15/FCS medium 100 ul of sheep anti-mouse IgG conjugated Dynabeads (Dynal) was added to the cells and incubated over ice for 20 min. After dilution in 10 ml L-15 medium, cells rosetted with Dynabeads were removed by adding the cells and medium to a 15 ml Falcon centrifuge tube in a Dynal MP-10 magnet and allowing beads to move to the side of the tube (1-2 min), following which the medium with unattached cells was withdrawn from the tube, and the process repeated using a fresh tube to ensure that all rosetted cells were removed. The remaining purified luminal cell suspension was then counted using a haemocytometer and the cells plated out at 2-5 million cells per 75-cm2 flask in RPMI1640/FCS medium. Attached cells were harvested for RNA within 3-4 days of replating.
2-3 days later the luminal-depleted primary cultures were treated again with Ca++-free medium overnight to detach residual luminal cells. These were discarded and the remaining attached cells, enriched for myoepithelial cells, were harvested by trypsinisation (1 mg/ml bovine pancreatic trypsin in 0.02% (w/v) EDTA for 5-10 min, after washing x2 in EDTA solution). Trypsinised cells were detached by tapping the flasks sharply and cells resuspended in L-15/10%FCS. Pelleted cells were resuspended in fresh L-15 and filtered through a 40-micron filter to remove clumps. The predominantly single cell suspension remaining was pelleted and resupended in 1 ml L-15/10%FCS, prior to adding a mouse IgG2a anti-CD10 monoclonal (SeraLab) and the IgG1 Ber-EP4 mouse monoclonal antibody (DAKO), both at 5 ug/ml, and incubating over ice, with rocking, for 1 hr. After washing, cells were labelled with 200 ul of rat anti-mouse IgG2a+b MACS MicroBeads for 20 min on ice and loaded onto a pre-washed BS column in the VarioMACS magnet. After washing to remove unattached cells, the column was removed from the magnet and retained cells flushed out. These were pelleted, resuspended in 1 ml L-15 plus 10% FCS and 100 ul of sheep anti-mouse IgG1-specific Dynabeads added for 20 min over ice. After separation of rosetted cells using the MP-10 magnet, the remaining purified myoepithelial cells were pelleted, counted and plated at 2-5 million cells/75-cm2 culture flask. Replated cells were harvested for RNA after 3-4 days. Reagents: Denaturing solution: 4 M Guanidinium thiocyanate, 25 mM Sodium citrate, 0.5% Sarcosyl, 0.1 M 2-mercaptoethanol; Solution D: prepare by adding 360 µl 2-mercaptoethanol to 50 ml stock guanidinium thiocyanate solution.
Procedure: 1. Pellet cells. Lyse cells in 100 µl Solution D per 1 million cells. (For a 10 cm2 monolayer culture of cells, add 1.8 ml Solution D)
2. For every 1 ml Solution D used, sequentially add: 100 µl 2 M Sodium acetate (pH 4.0), 1 ml Phenol, 200 µl Chloroform-isoamyl alcohol mix (49:1). Mix thoroughly after each addition.
3. Vigorously shake the final suspension for 10 seconds after the final addition and cool on ice for 15 minutes.
4. Centrifuge at 10,000g for 20 minutes at 4C.
5. After centrifugation, RNA is present in the aqueous phase, whereas DNA and proteins are present in the interphase and phenol phase.
6. Transfer the aqueous phase to a fresh tube and mix with 1 ml isopropanol per 1 ml of solution D used. Store at -20C for at least 1 hour to precipitate RNA.
7. Centrifuge at 10,000g for 20 minutes at 4C.
8. Dissolve RNA pellet in 300 µl Solution D.
9. Transfer to 1.5 ml Eppendorf tube and precipitate with 1 volume isopropanol at -20C for 1 hour.
10. Centrifuge at 10,000g for 10 minutes at 4C.
11. Wash the pellet in 75% ethanol and centrifuge at 10,000g for 10 minutes at 4C. Dry pellet and dissolve in DEPC-treated water or 1 mM EDTA, pH 8.0 (DEPC-treated).
12. Check RNA quantity and quality by conventional spectophotometry and formaldehyde/agarose gel electrophoresis, or on an Agilent BioAnalyser 2100. 1. 25 ug of total RNA was precipitated by adding 1/40th volume of 3 M sodium acetate (pH 5.2) to this RNA mixture which should already be in 75% ethanol. Allow precipitation to occur at -70C for 20-30 min.
2. Spin for 10 min at 12000x g in a microcentrifuge to pellet the RNA. Wash pellet briefly in 100 ul of 70% ethanol and air dry.
3. Resuspend RNA pellet in the following mixture: 12.9 ul DEPC ddH2O + 2.5 ul anchored oligo-dT17 (2 ug/ul).
4. Heat the RNA/oligo mixture to 70C for 10 min and snap-chill on ice.
5. Add the following reaction mix and incubate at 42C for 2 hours: 15.4 ul of RNA/oligo mix, 6.0 ul of 5x first strand buffer, 3.0 ul of 0.1 M DDT, 0.6 ul of dNTP mix (25 mM dATP, dTTP, dGTP, and 10 mM dCTP), 3.0 ul of dCTP-Cy5 (1 mM stock, Amersham PA53021, PA55021), 2.0 ul of Superscript II (200 U/ul); total volume: 30.0 ul.
6. Add 1.5 ul of 1 M NaOH and incubate at 70C for 20 min to hydrolyze the RNA.
7. Add 1.5 ul of 1 M HCl to neutralize the reaction.
8. Follow instructions for removal of nucleotides and short oligomers supplied with AutoSeq G-50 columns (Amersham Pharmacia BioTech, 27-5340-02). Once labeled ss cDNA has been purified through the G-50 columns the resulting flow-through should be approximately 33 ul in volume.
First strand buffer, DTT, and Superscript II are supplied by Invitrogen, cat.no.: 18064-014. 1. 25 ug of total RNA was precipitated by adding 1/40th volume of 3 M sodium acetate (pH 5.2) to this RNA mixture which should already be in 75% ethanol. Allow precipitation to occur at -70C for 20-30 min.
2. Spin for 10 min at 12000x g in a microcentrifuge to pellet the RNA. Wash pellet briefly in 100 ul of 70% ethanol and air dry.
3. Resuspend RNA pellet in the following mixture: 12.9 ul DEPC ddH2O + 2.5 ul anchored oligo-dT17 (2 ug/ul).
4. Heat the RNA/oligo mixture to 70C for 10 min and snap-chill on ice.
5. Add the following reaction mix and incubate at 42C for 2 hours: 15.4 ul of RNA/oligo mix, 6.0 ul of 5x first strand buffer, 3.0 ul of 0.1M DDT, 0.6 ul of dNTP mix (25 mM dATP, dTTP, dGTP, and 10 mM dCTP), 3.0 ul of dCTP-Cy3 (1 mM stock, Amersham PA53021, PA55021), 2.0 ul of Superscript II (200 U/ul); total volume: 30.0 ul.
6. Add 1.5 ul of 1 M NaOH and incubate at 70C for 20 min to hydrolyze the RNA.
7. Add 1.5 ul of 1 M HCl to neutralize the reaction.
8. Follow instructions for removal of nucleotides and short oligomers supplied with AutoSeq G-50 columns (Amersham Pharmacia BioTech, 27-5340-02). Once labeled ss cDNA has been purified through the G-50 columns the resulting flow-through should be approximately 33 ul in volume.
First strand buffer, DTT, and Superscript II are supplied by Invitrogen, cat.no.: 18064-014. 1. Combine the two ss cDNA samples as follows: 33 ul of ss cDNA sample 1 (Cy3-labeled), 33 ul of ss cDNA sample 2 (Cy5-labeled), 4 ul polyA DNA (2 ug/ul, Sigma, P0887), 4 ul of human Cot1 DNA (2 ug/ul; Gibco BRL, 15279-011), 7 ul of 3 M sodium acetate pH 5.2 and 218 ul of 100% ethanol (total volume ~293 ul).
2. Precipitate ss cDNAs, polyA and Cot1 DNA at -70 C for 20 min.
3. Pellet sample and wash briefly in 70% ethanol. Dry pellet thoroughly.
4. Resuspend pellet in 40 ul of hybridization buffer (5 x SSC, 6 x Denhardt's solution, 60 mM Tris HCl pH 7.6, 0.12% sarkosyl, 48% formamide; filter sterilized), and add 8 ul of ddH2O to bring the final volumne to 48 ul.
5. Place ss cDNA mixture in a 100C heat block or water bath for 5 min. Remove and allow to cool at room temperature for 10 min. Spin briefly in a microcentrifuge to remove the evaporated liquid from the lid of tube.
6. Carefully, apply the sample in hybridization buffer onto the center of the coverslip placed on a flat surface.
7. Invert the slide containing the microarray so that the DNA side is facing downward. Gently lower the slide onto the coverslip so that coverslip edge abuts the label of the array - this will ensure that the entire array is covered by the coverslip (although the coverslip may slightly overhang the other end of the slide). Once the hybridization mixture makes contact with the lowering slide, it will spread evenly across the microarray. Any small air bubbles will gradually disappear during the hybridization (but try to avoid making them if possible).
8. Place microarray slide DNA side up in a humid chamber (2 x 7 cm 3MM paper moistened with 2 ml of 40% formamide, 2 x SSC in a slide box) to prevent buffer from evaporating during hybridization.
9. Incubate at 47C for 12-24 hours.
10. Place microarray in 400 ml of room temperature wash solution 1 (2 x SSC, filter sterilized).
11. Transfer microarray slide in 50 ml Falcon tube to wash solution 2 (0.1 x SSC, 0.1% SDS, filter sterilized). Wash at room temperature for 15 min with vigorous shaking.
12. Repeat step 11.
13. Transfer microarray to wash solution 3 (0.1 x SSC, filter sterilized). Wash at room temperature for 5 min with vigorous shaking for 4 times.
14. Quickly transfer microarray in 50 ml Falcon tube to a centrifuge (Sorvall RT 6000D or equivalent) and spin at 1000 rpm for 1-2 min to dry the slides. The log expression ratios were normalized using lowess local regression (Yang et al., 2002) using the statistical platform S-Plus version 6.1 for Windows (Insightful). (Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai and T. P.Speed (2002). "Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation." Nucleic Acids Res 30(4): e15.
Formulae used: Lr <- logb(rg$R,2);Lg <- logb(rg$G,2); M <- Lr - Lg
Protocol Parameters min temperature;media;start Time;stop Time; media;min temperature;stop Time;start Time; start Time;media;stop Time;min temperature; Extracted product;Amplification; Amount of nucleic acid labeled;Label used;Amplification; Label used;Amplification;Amount of nucleic acid labeled; Quantity of label target used;Time;Chamber type;Volume;Temperature;
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF
SDRF File E-MEXP-36.sdrf.txt
Term Source Name mo ArrayExpress mo EFO
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version